Pharmaceutical compositions and methods to vaccinate against candidiasis

ABSTRACT

A  Candida albicans  bloodstream infections cause significant morbidity and mortality in hospitalized patients. Filament formation and adherence to host cells are critical virulence factors of  C. albicans . Multiple filamentation regulatory pathways have been discovered, however the downstream effectors of these regulatory pathways remain unknown. The cell surface proteins in the ALS group are downstream effectors of the filamentation regulatory pathway. Particularly, Als1p mediates adherence to endothelial cells in vitro and is required for virulence. The blocking of adherence by the organism is described resulting from the use of a composition and method disclosed herein. Specifically, a pharmaceutical composition comprised of a gene, gene product, or specific antibody to the ALS gene family is administered as a vaccine to generate an immune response capable of blocking adherence of the organism.

RELATED INFORMATION

This application is a continuation-in-part of Ser. No. 09/715,876 filedon Nov. 18, 2000, which is a priority from Provisional Application Ser.No. 60/166,663 filed Nov. 19, 1999.

This invention was made with Government support under Public HealthService grants PO-1AI-37194, RO1AI-19990, and MO1 RR0425. The Governmenthas certain rights in this invention. The priority of the priorapplications are expressly claimed, and the disclosure of each of theseprior applications are hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

This invention relates to Candida albicans surface adhesin proteins, toantibodies resulting from an immune response to vaccination, tocompositions used as prophylactic or therapeutic vaccines, and tomethods for the prevention and/or treatment of candidiasis.

BACKGROUND OF INVENTION

A dramatic increase in the incidence of nosocomial infections caused byCandida species has been observed in recent years. The incidence ofhematogenously disseminated candidal infections increased 11-fold from1980 to 1989. This increasing incidence has continued through the 1990sand into the 2000s. Infections by Candida species are now the fourthmost common cause of nosocomial septicemia, are equal to that ofEscherichia coli, and surpass the incidence caused by Klebsiellaspecies. Furthermore, Candida species are the most common cause ofdeep-seated fungal infections in patients who have extensive burns. Upto 11% of individuals undergoing bone marrow transplantation and 13% ofthose having an orthotopic liver transplant will develop an invasivecandidal infection.

C. albicans infections are difficult to diagnose and the organism cansurvive in vivo without causing overtly detectable disease symptoms andcan cause overt disease symptoms that vary in site and in severity. Theinfection can be localized and superficial or systemic and disseminated.C. albicans possesses numerous mechanisms to adapt to host sites, anddifferential gene expression under these mechanisms. Candida albicanscan switch between two morphologies: the blastospore (budding yeast) andfilamentous (hyphae and pseudohyphae) phases. Candida mutants that aredefective in genes regulating filamentation are reported to have reducedvirulence in animal models. This reduced virulence suggests that theability to change from a blastospore to a filament is a key virulencefactor of C. albicans. To date, no essential effectors of thesefilamentation pathways have been identified in C. albicans. SeeCaesar-TonThat, T. C. and J. E. Cutler, “A monoclonal antibody toCandida albicans enhances mouse neutrophil candidacidal activity,”Infect. Immun. 65:5354-5357, 1997.

The identification of effectors in the regulatory pathways of theorganism that contribute to virulence offers the opportunity fortherapeutic intervention with methods or compositions that are superiorto existing antifungal agents. The identification of cell surfaceproteins that effect a regulatory pathway involved in virulence isparticularly promising because characterization of the proteins enablesimmunotherapeutic techniques that are superior to existing antifungalagents when fighting a candidal infection. Also, both passive and activevaccination techniques offer unique prophylactic or therapeuticutilities depending on the clinical setting.

While potent antifungal agents exist that are microbicidal for Candida,the attributable mortality of candidemia is approximately 38%, even withtreatment with potent anti-fungal agents such as amphotericin B. Also,existing agents such as amphotericin B tend to exhibit undesirabletoxicity. Although additional antifungals may be developed that are lesstoxic than amphotericin B, it is unlikely that agents will be developedthat are more potent. Therefore, either passive or active immunotherapyto treat or prevent disseminated candidiasis is a promising alternativeto standard antifungal therapy.

The virulence of Candida albicans is regulated by several putativevirulence factors, of which adherence to host constituents and theability to transform from yeast-to-hyphae are among the most critical indetermining pathogenicity. Adherent strains of C. albicans are morevirulent than less-adhesive strains. Moreover, the more frequentlyisolated pathogenic species exhibit greater adhesive capacity.Investigations to understand C. albicans adhesion have involvedcharacterization of the cell surface, since this is the initial point ofcontact between fungus and host. Moreover, filmentation pathways andtheir effect on molecules and pathways are implicated in virulence.

SUMMARY OF INVENTION

The present invention utilizes the gene, the family of gene products, aspecific anisera of C. albicans agglutinin like sequence as a vaccine totreat, prevent, or alleviate disseminated candidiasis. The inventionincludes specially formulated compositions containing the ALS1apolynucleotides, the ALS 1a polypeptides, monoclonal and polyclonalantisera specifically reactive with these molecules and compositionscontaining forms or derivatives of any of the foregoing molecules suchas fragments or truncations. All of the compositions and methods of theinvention take advantage of the role of the group of the ALS1 geneproducts in the adherence of the C. albicans to endothelial andepithelial cells and the role of ALS expression in adherence andfilamentation and in the overall virulence of C. Albicans. Specifically,the control of ALS1 expression by transcription factor Efg1p, which isknown to be a regulation of filamentation, demonstrates thesusceptibility of the ALS1-expressed surface protein for use intherapeutic strategies, e.g. for use of the polypeptide as a vaccine toretard the pathogenesis of the organism, for use of antisera (polyclonalor monoclonal antibodies) in a passive immunization strategy, or forimmunization by polynucleotide vaccination.

Pursuant to this invention, a member of the ALS gene family encodes asurface adhesin that is selected as the target of an immunotherapeuticstrategy against Candida albicans. A demonstration that an expressionproduct of an ALS gene has structural characteristics typical of surfaceproteins and is, in fact, expressed on the cell surface of C. albicansis a critical first criterion for any member of the group of proteinsthat acts as an adhesin to host tissues. For example, ALS1p has a signalpeptide at the N-terminus, a glycosylphosphatidylinosine (GPI) anchoragesequence in the C-terminus, and a central region comprising repeats richin threonine and serin. N—, and O— as well as several glycosylationsites, which is typical of proteins that are expressed on the cellsurface. Indirect immunofluorescence using a monoclonal antibodydirected against the N-terminus of Als1p revealed that Als1p isexpressed during the log phase of blastospores. This expression of Als1pis increased during hyphal formation and is localized to the junctionwhere the hyphal element extends from the blastospores as indicated bythe diffused surface staining. Furthermore, a monoclonal antibodyblocked the enhanced adherence of C. albicans overexpression mutant toendothelial cells, thereby establishing the principle for immunotherapyapplications using members of the ALS family. The N-terminal region is aprime candidate for both passive and active immunization strategies andthe gene and gene product can be used in a full length, truncated ormodified form.

Additional evidence that ALS1p is a surface adhesin protein is based ondata showing that antibodies that bind to the surface of C. albicansalso bind to the surface of S. cerevisiae transformed with ALS1, but notwith empty plasmid. The ALS1 protein also shares significant homologywith the alpha-agglutinin of S. cerevisiae, which is expressed on thecell surface and mediates the binding of mating type alpha cells tomating type a cells. Moreover, expression of the ALS1 gene in S.cerevisiae increases the adherence of this organism to endothelial cellsby approximately 100-fold. Because the ALS1 gene appears to encode afunctional adhesin in Saccharomyes cerevisiae, it is certain that italso encodes a functional adhesin in C. albicans. The ALS1 gene wasoriginally isolated by Hoyer et al. without a known function. Hoyer, L.L., S. Scherer, A. R. Shatzman, and G. P. Livi. 1995. Candida albicansALSI: domains related to a Saccharonzyces cerevisiae sexual agglutininseparated the direct role of ALS1p in the various virulence pathwaysdescribed herein, e.g. adherence, filamentation, and floculation satisfya second criteria for use as a therapeutic agent, and lead to thetherapeutic embodiments of the invention as described in greater detailbelow. Recognition of the unique characteristics of the ALS1 geneproduct in the pathogenesis of the organism suggests several discretetherapeutic approaches that interrupt critical virulence factors orpathways by a repeating motif. Mol. Microbiol. 15:39-54. (See also U.S.Pat. Nos. 5,668,263 and 5,817,466.)

In addition to the administration of anti-fungal agents,immunotherapeutic therapies enabled by the invention are employed tofight a fungal infection as part of an integrated anti-fungal clinicalstrategy that combines traditional anti-fungal agents withimmunotherapeutics. Immunotherapeutics can be broadly defined in twocategories: active and passive. Active immunotherapy relies on theadministration of an antigenic compound as a vaccine that causes thebody's immune system to mount an immune response to the compound.Typically, the immune response includes cell-mediated immune pathwaysand the generation of antibodies against the antigenic compound. Inactive immunization, antibodies specific for the compound and generatedby the body also fight the fungal infection. Passive immunotherapyinvolves direct administration of the antibodies without the antigen. Inpassive immunization, the antibodies may be generated in vitro, such asin a conventional hybridoma or other expression system, and areadministered directly to a patient. Both active and passive immunizationoffer the advantage of using antibodies that are highly specific andtypically far less toxic than ordinary anti-fungal agents.

Although certain data and results presented herein are specific to theALS1 species and related compounds, additional members of the ALS familyexhibit similar functionality in the pertinent virulence pathways ofCandida. The other members of the family, generally designated asALS1-ALS1a share significant sequence homology with ALS1 and with eachother and show highly conserved regions N terminal region. See FIG. 7.Thus, according to one aspect of the invention, a member of the ALSsurface adhesin family of proteins or a fragment, conjugate, or analoguethereof, is formulated in a pharmaceutical composition and administeredas a vaccine. ALS surface adhesin proteins are preferably obtained fromCandida albicans, however, similar adhesin molecules or analogues orderivatives thereof may be of candidal origin and may be obtainable fromstrains belonging to the genera Candida such as Candida parapsilosis,Candida Candida krusei, Candida dublinoensis, and Candida tropicalis. Asurface adhesin protein according to the invention may be obtained inpurified form, and thus, according to a preferred embodiment of theinvention, a substantially pure ALS Candida albicans surface adhesinprotein, or functional analogue, conjugate, or derivative thereof, isformulated as a vaccine to cause an immune response in a patient toblock adhesion of the organism to the endothelial cells.

An analogue or derivative of the surface adhesion protein according tothe invention may be identified and further characterized by thecriteria described herein for the ALS gene and gene product. Forexample, a null mutant of the analogue or derivative would sharemarkedly reduced adhesion to endothelial cells compared to controls.Similarly, over-expression of the analogue or derivative in anappropriate model would show an increased adherence to endothelial cellscompared to controls and would be confirmed as a cell surface adhesin inaccord with the criteria described above. Also, antisera to the analogueor derivative would cross-react with anti-ALS antibodies and would alsoexhibit increased survival times when administered in animal models ofdisseminated candidiasis as disclosed herein.

The present invention also provides an immunotherapeutic strategyagainst Candida infection at the level of binding to the vascularendothelial cells and through a downstream effector of the filamentationregulatory pathway. An immunotherapeutic strategy is uniquelyadvantageous in this context because: (i) the morbidity and mortalityassociated with hematogenously disseminated candidiasis remainsunacceptably high, even with currently available antifungal therapy;(ii) a rising incidence of antifungal resistance is associated with theincreasing use of antifungal agents, (iii) the population of patients atrisk for serious Candida infections is well-defined and very large, andincludes post-operative patients, transplant patients, cancer patientsand low birth weight infants; and (iv) a high percentage of the patientswho develop serious Candida infections are not neutropenic, and thus mayrespond to a vaccine. For these reasons, Candida is the most attractivefungal target for either passive or active immunotherapy.

Having determined the immunotherapeutic potential of members of the ALSfamily according to this invention, the gene, the protein gene product,conjugates, analogues, or derivative molecules thereof, and compositionscontaining specific monoclonal or polyclonal antisera may be used intreatment and/or prevention of candidal infections. Standardimmunological techniques may be employed with the adhesion proteinmolecule, and its analogues, conjugates, or derivatives, to use themolecule as an immunogen in a pharmaceutically acceptable compositionadministered as a vaccine. For the purposes of this invention,“pharmaceutical” or “pharmaceutically acceptable” compositions areformulated by known techniques to be non-toxic and, when desired, usedwith carriers or additives that are approved for administration tohumans in, for example, intravenous, intramuscular, intraperitoneal orsub-cutaneous injection. Such compositions may include buffers, salts orother solvents known to these skilled in the art to preserve theactivity of the vaccine in solution.

With respect to the molecule used as the immunogen pursuant to thepresent invention, those of skill in the art will recognize that eachprotein molecule within the ALS family may be truncated or fragmentedwithout losing the essential qualities as a vaccine. For example, theAls1p may be truncated to yield an N-terminal fragment by truncationfrom the C-terminal end with preservation of the functional propertiesdescribed above and may include all or a portion of the GP1 anchorsequences on the central region. Likewise, C-terminal fragments may becreated by truncation from the N-terminal end with preservation of thefunctional properties described above. Other modifications in accordwith the foregoing rationale may be made pursuant to this invention tocreate other ALS protein analogs or derivatives, to achieve the benefitsdescribed herein with the native protein.

The goal of the immunotherapy provided by this invention is to interferewith regulation of filamentation, to block adherence of the organism tohost constituents, and to enhance clearance of the organism byimmunoeffector cells. Since endothelial cells cover the majority of thevasculature, specially selected strategies, compositions, andformulations to block the adherence of the organism to endothelial cellsusing antibodies are a preferred embodiment of the present invention andsuch adherence blocking strategies include active or passiveimmunotherapy directed against the candidal adhesin(s) disclosed herein.Specific anti-sera having demonstrated abilities to interrupt virulencefactors and pathways implicated in virulence are identified herein basedon the identification of the unique properties of the ALS family ofproteins and specific derivatives thereof, including N-terminalfragments of the ALS proteins, specific monoclonal and polyclonalantisera against regions of the protein molecule, and polynucleotidesselectively encoding this region.

Depending on the specific virulence of a strain in a clinical setting, apharmaceutical composition comprising either monoclonal or polyclonalantibodies may be administered in a passive immunization therapy.Polyclonal antibodies are thought to involve fewer specific crossreactivity reactions that may lead to acute toxicity, whereas monoclonalantibodies provide more reproducible binding to a specific epitope of atarget protein. Therefore, selection of the species for passiveimmunotherapy depends on the specific organism encoding theprotein-antigen, as well as the specific antibody raised against the ALSprotein. In either case, the antisera is specific to a portion of theALS protein and functions to interrupt virulence regulatory pathwaysnecessary for pathogenesis of the organism. Specifically, the antiseraaffects the Efglp filamentation pathway and expression of the surfaceprotein implicated in both floculation and adherence to endothelialcells. Characteristic antisera of the invention interrupt the role ofALS in filamentation and virulence mechanisms in both in vitro systemsas well as animal models of disseminated candidiasis.

The method of the invention also includes ameliorating and/or preventingcandidal infection by blocking the adherence of C. albicans to theendothelial cells of a host constituent. Thus, according to one aspectof the invention, a pharmaceutical composition comprising an ALS adhesinprotein derivative, analogue, or conjugate is formulated as a vaccine ina pharmaceutical composition containing a biocompatible carrier forinjection or infusion and is administered to a patient. Prior toinjection, the adhesin protein may be formulated as a vaccine in asuitable vehicle, preferably a known immunostimulant such as apolysaccharide. Thus, according to a further aspect of the invention weprovide a pharmaceutical composition comprising a candidal adhesinprotein together with one or more pharmaceutically acceptable excipientsin a formulation for use as a vaccine. Also, direct administration ofantiserum raised against an ALS protein may be used as a therapeutic orprophylactic strategy to block the adherence of C. albicans to amammalian host constituent. Thus, for example, any suitable host may beinjected with protein and the serum collected to yield the desiredanti-adhesin antibody after appropriate purification and/orconcentration. Monoclonal antiserum against adhesin protein can beobtained by known techniques, Kohler and Milstein, Nature 256: 495-499(1975), and may be humanized to reduce antigenicity, see U.S. Pat. No.5,693,762, or produced by immunization of transgenic mice having anunrearranged human immunoglobulin gene, see U.S. Pat. No. 5,877,397, toyield high affinity (e.g. 10⁸, 10⁹, or 10¹⁰) anti-ALS IgG monoclonalantibodies.

A still further use of the invention, for example, is using an ALSadhesin protein to develop a specific clinical vaccine strategies forthe prevention and/or amelioration of candidal infections. Thus,according to one aspect of the invention, for example, standardimmunology techniques may be employed to construct a multi-protein orprotein fragment component vaccine strategy that may enhance and/orelicit immune response from a host constituent to bock adherence of C.albicans. Also, known immunostimulatory compositions may be added to thevaccine formulation, wherein such compounds include known proteins,saccharides or oligonucleotides. (See Krieg U.S. Pat. No. 6,008,200).

A still further use of the invention, for example, is an isolatedpolynucleotide, RNA or DNA vaccine strategy wherein the ALSpolynucleotide encoding an ALS protein or a fragment or variant thereofis administered according to a protocol designed to yield an immuneresponse to the gene product. See e.g., Feigner U.S. Pat. No. 5,703,055.Generally, the naked polynucleotide is combined in a pharmaceuticallyacceptable injectable carrier and injected into muscle tissue where thepolynucleotide is transported into cells and expressed to produce aselectively induced immunogenic response comprised of antibodies againstthe polypeptide encoded by the polynucleotide. The tissue into which thepolynucleotide is introduced is preferably muscle, but can be any tissuethat expresses the polynucleotide. The polynucleotide may be either aDNA or an RNA sequence and when the DNA is used, the DNA sequence can beinserted into a plasmid that also contains a replicator. In thisembodiment, a method of immunization is provided by obtaining anexpressible polynucleotide coding for an immunogenic ALS polypeptide,and introducing the polynucleotide into a patient to elicit expressionof the ALS polypeptide and the generation of an immune response againstthe immunogen such that an anti-ALS antibody composition produced invivo provides protection against Candidiasis by disrupting the virulencepathway, for example, as has been associated with ALS1p and the effectorpathway for adhesion and filamentation of the Candida organism.Particularly preferred polynucleotide compositions encode N-terminalregions of an ALS polypeptide and code for the specific regions thatelicit the antisera production in vivo that are shown herein to exhibitthe prophylactic therapeutic utility derived from interruption ofCandida virulence mechanisms.

A still further use of the invention, for example, is developingcombination vaccine strategies. Thus, according to one aspect of theinvention, for example, anti-ALS antibodies may be used with antibodiesin treating and/or preventing candidal infections. See U.S. Pat. No.5,578,309.

DESCRIPTION OF THE FIGURES

FIG. 1A, 1B show the mediation of Als1p adherence of C. albicans tohuman umbilical vein endothelial cells. Values represent the mean±SD ofat least three independent experiments, each performed in triplicate.(A) Endothelial cell adherence of ALS1/als2, als1/als1 andALS1-complemented mutants and wild-type CAI12 (30) (B) Endothelial celladherence of P_(ADH1)-ALS1 mutant that overexpresses ALS1, compared towild type C. albicans. Statistical treatment was obtained by Wilcoxonrank sum test and corrected for multiple comparisons with the Bonferronicorrection. *P<0.001 for all comparisons.

FIG. 2A-D shows the cell surface localization of Als1p on filaments ofC. albicans by indirect immunofluorescence. Filamentation of C. albicanswas induced by incubating yeast cells in RPMI 1640 medium with glutaminefor 1.5 hours at 37° C. Als1p was detected by incubating organisms firstwith anti-Als1p mouse mAb followed by FITC-labeled goat anti-mouse IgG.C. albicans cell surface was also stained with anti-C. albicanspolyclonal Ab conjugated with Alexa 594 (Molecular Probes, Eugene,Oreg.). Areas with yellow staining represent Als1p localization. (A) C.albicans wild-type. (B) als1/als1 mutant strain. (C) als1/als1complemented with wild type ALS1 (D)P_(ADH1)-ALS1 overexpression mutant.

FIG. 3A, 3B show the mediation of Als1p on C. albicans filamentation onsolid medium. C. albicans blastospores were spotted on Lee's agar platesand incubated at 37° C. for 4 days (A) or 3 days (B).

FIG. 4A, 4B show the control of ALS1 expression and the mediation of C.albicans filamentation by the EFG1 filamentation regulatory pathway. (A)Northern blot analysis showing expression of ALS1 in (i) mutantsdeficient in different filamentation regulatory pathways. (ii) efg1/efg1mutant complemented with either EFG1 or P_(ADH1)-ALS1. Total RNA wasextracted from cells grown in RPM1 1640+glutamine medium at 37° C. for90 minutes to induce filamentation. Blots were probed with ALS1 andTEF1. (B) Photomicrographs of the efg1/efg1 mutant and efg1/efg1 mutantcomplemented with P_(ADH1)-ALS1 grown on Lee's agar plates at 37° C. for4 days.

FIG. 5A, 5B show the reduction of virulence in the mouse model ofhematogenously disseminated candidiasis by (A) Male Balb/C mice (n=30for each yeast strain) were injected with stationary phase blastospores(10⁶ per mouse in 0.5 ml of PBS). Curves are the compiled results ofthree replicate experiments (n=30 mice for each strain). The doublingtimes of all strains, grown in YPD at 30° C., ranged between 1.29 to1.52 hours and were not statistically different from each other.Southern blot analysis of total chromosomal DNA was used to match theidentity of the genotype of C. albicans strains retrieved from infectedorgans with those of C. albicans strains used to infect the mice.Statistical analysis was obtained by Wilcoxon rank sum test andcorrected for multiple comparisons with the Bonferroni correction.*P<0.002 for the als1/als1 mutant versus each of the other strains. (B)Histological micrographs of kidneys infected with C. albicans wild-type,homozygous als1 null mutant, or heterozygous ALS1 complemented mutant.Kidney samples were retrieved 28 hours (a) or 40 (b) hours postinfection, fixed in paraformaldehyde and sections were stained withsilver (magnification, X400). Arrows denote C. albicans cells.

FIG. 6 shows the prophylactic effect of anti-ALS antibody againstdisseminated candidiasis as a function of surviving animals over a30-day period for animals infused with anti-Als1p polyserum.

FIG. 7 is the protein sequence alignment of the N-terminal portion ofselect ALS proteins arranged by adherence phenotype. The top three linesthe sequences from ALS proteins that bind endothelial cells, and thebottom three are sequences from ALS proteins that do not bindendothelial cells. Boxes represent areas of significant sequencedivergence that are candidate substrate binding domains.

DETAILED DESCRIPTION OF THE INVENTION

The nature of the pathogenesis of C. albicans by adherence toendothelial cells is discussed in U.S. Pat. No. 5,578,309 which isspecifically incorporated herein by reference in its entirety. For adescription of the ALS1 gene and characteristics thereof, including thecharacterization of the gene product as an adhesin, see Fu, Y., S. G.Filler, B. J. Spellberg, W. Fonzi, A. S. Ibrahim, T. Kanbe, M. A.Ghannoum, and J. E. J. Edwards. 1998. Cloning and characterization ofCAD I/AAF1, a gene from Candida albicans that induces adherence toendothelial cells after expression in Saccharonzyces cerevisiae. Infect.Immun. 66:2078-2084; Fu, Y., G. Rieg, W. A. Forizi, P. H. Belanger, J.E. J. Edwards, and S. G. Filler. 1998. Expression of the Candidaalbicans gene ALS1 in Saccharomyces cerevisiae induces adherence toendothelial and epithelial cells. Infect. Immun. 66:1783-1786; Hoyer, L.L. 1997. The ALS gene family of Candida albicans. International Societyfor Human and Animal Mycology Salsimorge, Italy:(Abstract); Hoyer, L.L., S. Scherer, A. R. Shatzman, and G. P. Livi. 1995. Candida albicansALSI: domains related to a Saccharonzyces cerevisiae sexual agglutininseparated by a repeating motif. Mol. Microbiol. 15:39-54. Thepolynucleotide sequence of the ALS1 gene and protein are SEQ ID NO:7 andNO:8, respectively. The remaining numbers of the ALS family of gene andprotein ALS-2-ALS-9, are SEQ ID NO:9-SEQ ID. NO:24. Note that the formsometimes known as ALS-N is ALS-9 and ALA-1 is ALS-5.

The following Examples illustrate the immunotherapeutic utility of theclass of ALS protein molecules as the basis for prevention or treatmentof disseminated candidiasis. Example 1 describes the preparation of anALS1 null mutant and a strain of C. albicans characterized byover-expression of ALS1 to confirm the mediation of adherence toendothelial cells. Example 2 describes the localization of Als1p and theimplication of the efg filamentation regulatory pathway. Example 3describes the purification of ALS1 adhesin protein. Example 4 describesthe preparation of antibodies raised against the ALS1 surface adhesinprotein to be used to demonstrate the blocking of the surface adhesinprotein. Example 5, describes the blocking of adherence in vivo, usingboth polyclonal and monoclonal antibodies raised against the ALS1surface adhesion protein as described herein to protect againstdisseminated candidiasis in a mouse model. Example 6 describes apolynucleotide vaccination strategy to cause in vivo expression of anantigenic ALS1p polypeptide to create a protective immune response.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are described. As usedherein, the following terms have the meanings ascribed to them unlessspecified otherwise.

“Polynucleotide” refers to a polymer composed of nucleotide units(ribonucleotides, deoxyribonucleotides, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof) linked via phosphodiester bonds, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof. Thus, the term includes nucleotide polymers in which thenucleotides and the linkages between them include non-naturallyoccurring synthetic analogs, such as, for example and withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs), and the like. Such polynucleotides can be synthesized, forexample, using an automated DNA synthesizer. The term “gene” typicallyrefers to a large number of polynucleotides that form a singlefunctional unit that is translated and transcribed to express apolypeptide of sufficient length to be immunogenic.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

“Antibody” or “antisera” refers to a polypeptide substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereof,which specifically bind and recognize an immunogen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Antibodies exist, e.g., as intactimmunoglobulins or as a number of well characterized fragments producedby digestion with various peptidases. This includes, e.g., Fab′ andF(ab)′.sub.2 fragments. The term “antibody,” as used herein, alsoincludes antibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

An antibody “is specific” or “specifically binds” to a protein when theantibody functions in a binding reaction which is determinative of thepresence of the protein in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind preferentially to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to a protein under suchconditions requires an antibody that is selected for its specificity fora particular protein. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

“Substantially pure” means an object species is the predominant speciespresent (i.e., on a molar basis, more abundant than any other individualmacromolecular species in the composition), and a substantially purifiedfraction is a composition wherein the object species comprises at leastabout 50% (on a molar basis) of all macromolecular species present.Generally, a substantially pure composition means that about 80% to 90%or more of the macromolecular species present in the composition is thepurified species of interest. The object species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) if the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), stabilizers (e.g., BSA), andelemental ion species are not considered macromolecular species forpurposes of this definition.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a mammal. A pharmaceutical composition comprises apharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effective” orphamaceutically effective” amount refers to that amount of an agenteffective to produce the intended pharmacological result.“Pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers, buffers, and excipients, such as a phosphatebuffered saline solution, 5% aqueous solution of dextrose, andemulsions, such as an oil/water or water/oil emulsion, and various typesof wetting agents and/or adjuvants. Suitable pharmaceutical carriers andformulations are described in Remington's Pharmaceutical Sciences, 19thEd. (Mack Publishing Co., Easton, 1995). Preferred pharmaceuticalcarriers depend upon the intended mode of administration of the activeagent. Typical modes of administration include enteral (e.g., oral) orparenteral (e.g., subcutaneous, intramuscular, or intravenousintraperitoneal injection; or topical, transdermal, or transmucosaladministration).

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit overt symptoms or signs of a disease or exhibits onlyearly signs for the purpose of decreasing the risk of developingpathology.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

Example 1 Als1 Mediates Adherence of C. albicans to Endothelial Cells

The URA blaster technique was used to construct a null mutant of Calbicans that lacks expression of the Als1p. The als1/als1 mutant wasconstructed in C. albicans strain CAI4 using a modification of theUra-blaster methodology [W. A. Fonzi and M. Y. Irwin, Genetics 134, 717(1993)] as follows: Two separate als1-hisG-IRA3-hisG-als1 constructswere utilized to disrupt the two different alleles of the gene. A 4.9 kbALS1 coding sequence was generated with high fidelity PCR (BoehringerMannheim, Indianapolis, Ind.) using the primers:5′-CCGCTCGAGATGCTTCAACAATTTACATTGTTA-3′ (SEQ ID NO.1) and5′-CCGCTCGAGTCACTAAATGAACAAGGACAATA3′ (SEQ ID NO. 2). Next, the PCRfragment was cloned into pGEM-T vector (Promega, Madison, Wis.), thusobtaining pGEM-T-ALS1. The hisG-URA3-hisG construct was released frompMG-7 by digestion with KpnI and Hind3 and used to replace the portionof ALS1 released by Kpn1 and Hind3 digestion of pGEM-T-ALS1. The finalals1-hisG-URA3-hisG-als1 construct was released from the plasmid bydigestion with Xhol and used to disrupt the first allele of ALS1 bytransformation of strain CAI-4.

A second als1-hisG-URA3-hisG-als1 construct was generated in two steps.First, a Bgl2-Hind3 hisG-URA3-hisG fragment of pMB7 was cloned into theBamH1-Hind3 sites of pUC19, thereby generating pYC2. PYC2 was thendigested with Hind3, partially filled in with dATP and dGTP using T4 DNApolymerase, and then digested with Sinai to produce a new hisG-URA3-hisGfragment. Second, to generate ALS1 complementary flanking regions,pGEM-T-ALS1 was digested with Xbal and then partially filled in withdCTP and dTTP. This fragment was digested with HpaI to delete thecentral portion of ALS1 and then ligated to the hisG-URA3-hisG fragmentgenerating pYC3. This plasmid was then digested by Xhol to release aconstruct that was used to disrupt the second allele of the ALS1. Growthcurves were done throughout the experiment to ensure that the generatedmutations had no effect on growth rates. All integrations were confirmedby Southern blot analysis using a 0.9 kb ALS1 specific probe generatedby digestion of pYF5 with XbaI and HindIII.

The null mutant was compared to C. albicans CAI-12 (a URA+revertantstrain) for its ability to adhere in vitro to human umbilical veinendothelial cells. For adherence studies, yeast cells from YPD (2%glucose, 2% peptone, and 1% yeast extract) overnight culture, were grownin RPMI with glutamine at 25° C. for 1 hour to induce Als1p expression.3×10² organisms in Hanks balanced salt solution (HBSS) (IrvineScientific, Irvine, Calif.) were added to each well of endothelialcells, after which the plate was incubated at 37° C. for 30 minutes. Theinoculum size was confirmed by quantitative culturing in YPD agar. Atthe end of incubation period, the nonadherent organisms were aspiratedand the endothelial cell monolayers were rinsed twice with HBSS in astandardized manner. The wells were over laid with YPD agar and thenumber of adherent organisms were determined by colony counting.Statistical treatment was obtained by Wilcoxon rank sum test andcorrected for multiple comparisons with the Bonferroni correction.P<0.001.

Referring to FIG. 1, a comparison of the ALS1/ALS1 and als1/als1 strainshowed that the ALS1 null mutant was 35% less adherent to endothelialcells than C. albicans CAI-12. To reduce background adherence, theadherence of the wild-type strain grown under non-ALS1 expressingconditions was compared with a mutant autonomously expressing Als1p.This mutant was constructed by integrating a third copy of ALS1 underthe control of the constitutive ADH1 promoter into the wild-type C.albicans. To achieve constitutive expression of the ALS1 in C. albicans,a blunt-ended PCR generated URA3 gene is ligated into a blunt-edged Bg12site of pOCUS-2 vector (Novagen, Madison, Wis.), yielding pOU-2. A 2.4kb Not1-Stu1 fragment, which contained C. albicans alcohol dehydrogenasegene (ADH1) promoter and terminator (isolated from pLH-ADHpt, and kindlyprovided by A. Brown, Aberdeen, UK), was cloned into pOU-2 afterdigestion with Not1 and Stul. The new plasmid, named pOAU-3 had only oneBg12 site between the ADH1 promoter and terminator. ALS1 coding sequenceflanked by BamH1 restriction enzyme sites was generated by high fidelityPCR using pYF-5 as a template and the following primers:5′-CGGGATCCAGATGCTTCA-ACAATTTACATTG-3′ (SEQ ID NO.3) and5′-CGGGATCCTCACTAAATGAACAAGGACAATA-3′ (SEQ ID NO.4). This PCR fragmentwas digested with BamH1 and then cloned into the compatible Bg12 site ofpOAU-3 to generate pAU-1. Finally, pAU-1 was linearized by XbaI prior totransforming C. albicans CAI-4. The site-directed integration wasconfirmed by Southern Blot analysis.

Referring to FIG. 1B, overexpressing ALS1 in this P_(ADH1)-ALS1 strainresulted in a 76% increase in adherence to endothelial cells, comparedto the wild-type C. albicans. In comparing endothelial cell adherence ofthe wild-type to that of the overexpressing mutant, yeast cells weregrown overnight in YPD at 25° C. (non-inducing condition of Als1p).Als1p expression was not induced to reduce the background adherence ofthe wild-type, thus magnifying the role of Als1p in adherence throughP_(ADH1)-ALS1 hybrid gene. The adherence assay was carried out asdescribed above. Statistical treatment was obtained by Wilcoxon rank sumtest and corrected for multiple comparisons with the Bonferronicorrection. P<0.001.

A monoclonal anti-Als1p murine IgG antibody was raised against apurified and truncated N-terminus of Als1p (amino acid #17 to #432)expressed using Clontech YEXpress™ Yeast Expression System (Palo Alto,Calif.). The adherence blocking capability of these monoclonalanti-Als1p antibodies was assessed by incubating C. albicans. cells witheither anti-Als1 antibodies or mouse IgG (Sigma, St. Louis, Mo.) at a1:50 dilution. After which the yeast cells were used in the adherenceassay as described above. Statistical treatment was obtained by Wilcoxonrank sum test and corrected for multiple comparisons with the Bonferronicorrection. P<0.001. The results revealed that the adherence of theP_(ADH1)-ALS1 strain was reduced from 26.8%+3.5% to 14.7%+5.3%. Thus,the effects of ALS1 deletion and overexpression demonstrate that Als1pmediates adherence of C. albicans to endothelial cells.

Example 2 Localization of Als1p

For a number of the ALS family to function as an adhesin protein, itmust be located on the cell surface. The cell surface localization ofAls1p, for example, was verified using indirect immunofluorescence withthe anti-Als1p monoclonal antibody. Diffuse staining was detected on thesurface of blastospores during exponential growth. This staining wasundetectable on blastospores in the stationary phase. Referring to FIG.2A, when blastospores were induced to produce filaments, intensestaining was observed that localized exclusively to the base of theemerging filament. No immunofluorescence was observed with the als1/als1mutant, confirming the specificity of this antibody for Als1p. See FIG.2B. These results establish that Als1p is a cell surface protein.

The specific localization of Als1p to the blastospore-filament junctionimplicates Als1p in the filamentation process. To determine themechanism, the filamentation phenotype of the C. albicans ALS1 mutantswas analyzed. Referring to FIG. 3A, the als1/als1 mutant failed to formfilaments after a 4 day incubation on Lee's solid medium, while both theALS1s/ALS1 and ALS1/als1 strains as well as the ALS1-complemented mutantproduced abundant filaments at this time point. The als1/als1 mutant wascapable of forming filaments after longer periods of incubation.Furthermore, overexpressing ALS1 augmented filamentation: theP_(ADH1)-ALS1 strain formed profuse filaments after a 3 day incubation,whereas the wild-type strain produced scant filaments at this timepoint. See FIG. 3B. To further confirm the role of Als1p infilamentation, a negative control was provided using mutant similar tothe ALS1 overexpression mutant, except the coding sequence of the ALS1was inserted in the opposite orientation. The filamentation phenotype ofthe resulting strain was shown to be similar to that of the wild-typestrain. The filament-inducing properties of Als1p are specific to cellsgrown on solid media, because all of the strains described abovefilamented comparably in liquid media. The data demonstrates that Als1ppromotes filamentation and implicates ALS1 expression in the regulationof filamentation control pathways. Northern blot analysis of ALS1expression in mutants with defects in each of these pathways, includingefg1/efg1, cph1/cph1, efg1/efg cph1/cph1, tup1/tup1, and cla4/cla4mutants were performed. Referring to FIG. 4A, mutants in which bothalleles of EFG1 had been disrupted failed to express ALS1. Introductionof a copy of wild-type EFG1 into the efg1/efg1 mutant restored ALS1expression, though at a reduced level. See FIG. 4B. Also, as seen inFIG. 4A, none of the other filamentation regulatory mutationssignificantly altered ALS1 expression (FIG. 4A). Thus, Efg1p is requiredfor ALS1 expression.

If Efg1p stimulates the expression of ALS1, which in turn inducesfilamentation, the expression of ALS1 in the efg1/efg1 strain shouldrestore filamentation. A functional allele of ALS1 under the control ofthe ADH1 promoter was integrated into the efg1/efg1 strain. Toinvestigate the possibility that ALS1 gene product might complement thefilamentation defect in efg1 null mutant, an Ura efg1 null mutant wastransformed with linearized pAU-1. Ura⁺ clones were selected andintegration of the third copy of ALS1 was confirmed with PCR using theprimers: 5′-CCGTTTATACCATCCAAATC-3′(SEQ ID NO. 5) and5′-CTACATCCTCCAATGATATAAC-3′ (SEQ ID NO.6). The resulting strainexpressed ALS1 autonomously and regained the ability to filament onLee's agar. See FIGS. 4B and C. Therefore, Efg1p induces filamentationthrough activation of ALS1 expression.

Because filamentation is a critical virulence factor in C. albicans,delineation of a pathway that regulates filamentation has importantimplications for pathogenicity. Prior to ALS1, no gene encoding adownstream effector of these regulatory pathways had been identified.Disruption of two other genes encoding cell surface proteins, HWP1ANDINTI, results in mutants with filamentation defects. Although HWP1expression is also regulated by Efg1p, the autonomous expression of HWP1in the efg1/efg1 mutant fails to restore filamentation. Therefore Hwp1palone does not function as an effector of filamentation downstream ofEFG1. Also, the regulatory elements controlling INT1 expression are notknown. Thus, Als1p is the first cell-surface protein identified thatfunctions as a downstream effector of filamentation, thereby suggestinga pivotal role for this protein in the virulence of C. albicans.

The contribution of Als1p to C. albicans virulence was tested in a modelof hematogenously disseminated candidiasis, A. S. Ibrahim et al.,Infect. Immun. 63, 1993 (1995). Referring to FIG. 5A, mice infected withthe als1/als1 null mutant survived significantly longer than miceinfected with the ALS1/ALS1 strain, the ALS1/als1 mutant or theALS1-complemented mutant. After 28 hours of infection, the kidneys ofmice infected with the als1/als1 mutant contained significantly fewerorganisms (5.70±0.46 log₁₀ CFU/g) (P<0.0006 for both comparisons). Nodifference was detected in colony counts of organisms recovered fromspleen, lungs, or liver of mice infected with either of the strains atany of the tested time points. These results indicate thatimmunotherapeutic strategies using ALS proteins as a vaccine have aprotective prophylactic effect against disseminated candidiasis. See SEQID NOS. 10, 12, 14, 16, 18, 20, 22, and 24. Referring to FIG. 5B,examination of the kidneys of mice after 28 hours of infection revealedthat the als1/als1 mutant produced significantly shorter filaments andelicited a weaker inflammatory response than did either the wild-type ofALS1-complemented strains. However, by 40 hours of infection, the lengthof the filaments and the number of leukocytes surrounding them weresimilar for all three strains.

The filamentation defect of the als1/als1 mutant seen on histopathologyparalleled the in vitro filamentation assays on solid media. This mutantshowed defective filamentation at early time points both in vivo and invitro. This defect eventually resolved with prolongedinfection/incubation. These results suggest that a filamentationregulatory pathway that is independent of ALS1 may become operative atlater time points. The activation of this alternative filamentationpathway by 40 hours of infection is likely the reason why mice infectedwith the als1/als1 mutant subsequently succumbed in the ensuing 2-3days.

Collectively, these data demonstrate that C. albicans ALS1 encodes acell surface protein that mediates both adherence to endothelial cellsand filamentation. Als1p is the only identified downstream effector ofany known filamentation regulatory pathway in C. albicans. Additionally,Als1p contributes to virulence in hematogenous candidal infection. Thecell surface location and dual functionality of Als1p make it anattractive target for both drug and immune-based therapies.

Example 3 Purification of ALS1 Adhesin Protein, Truncated N-TerminalProtein

For use as an immunogen, an ALS protein synthesized by E. coli isadequate when vaccination with a traditional protocol yield an immuneresponse generating B cells expressing measurable anti-ALS anti-sera orlevels of serum Ig from which polyclonals may be obtained. However,eukaryotic proteins synthesized by E. coli may not be functional due toimproper folding or lack of glycosylation. Therefore, to determine ifthe ALS1 protein can block the adherence of C. albicans to endothelialcells, the protein is, preferably, purified from genetically engineeredC. albicans, and formulated into a substantially pure pharmaceuticalcomposition that is pharmacologically effective for prophylactic ortherapeutic treatment of disseminated candidiasis.

PCR was used to amplify a fragment of ALS1, from nucleotides 52 to 1296.This 1246 by fragment encompassed the N-terminus of the predicted ALS1protein from the end of the signal peptide to the beginning of thetandem repeats. This region of ALS1 was amplified because it likelyencodes the binding site of the adhesin, based on its homology to thebinding region of the S. cerevisiae Agα1 gene product. In addition, thisportion of the predicted ALS1 protein has few glycosylation sites andits size is appropriate for efficient expression in E. coli.

The N-terminal fragment of ALS1 was ligated into pQE32 to produce pINS5.In this plasmid, the N-terminal segment of the protein is expressedunder control of the lac promoter and it has a 6-hits tag fused to itsN-terminus so that it can be affinity purified. We transformed E. coliwith pINS5, grew it under inducing conditions (in the presence of IPTG),and then lysed the cells. The cell lysate was passed through aNi²⁺-agarose column to affinity purify the ALS1-6His fusion protein.This procedure yielded substantial amounts of ALSI-6His. The fusionprotein was further purified by SDS-PAGE. The band containing theprotein was excised from the gel so that antiserum can be raised againstit as described in detail herein. It will be appreciated by one skilledin the art that the surface adhesin protein according to the inventionmay be prepared and purified by a variety of known processes withoutdeparting from the spirit of the present invention based on thepolynucleotide and polypeptide sequences of listed in SEQ ID. NO.1-SEQID NO.18. As noted above, analogues and derivatives of ALS1p may beprepared by known techniques based on conserved principles of amino acidsubstitution and nucleotide encoding degeneracy without departing fromthe invention. Thus, Such compositions may exhibit at least oneconservative substitution in the polypeptide sequence and exhibit thesame effect in disruption of adherence and filamentation pathways as thenative ALS1p and antibodies that specifically bind thereto as describedherein.

Example 4 Raising Polyclonal Antisera Against ALS1 Protein

To determine whether antibodies against the ALS1 protein block theadherence of Candida albicans to endothelial and epithelial cells, andthe selected host constituent in vitro, rabbits were inoculated with S.cerevisiae transformed with ALS1 protein. The immunization protocol usedwas the dose and schedule used by Hasenclever and Mitchell forproduction of antisera that identified the antigenic relationship amongvarious species of Candida. Hasenclever, H. F. and W. O. Mitchell. 1960.Antigenic relationships of Torulopsis glabrata and seven species of thegenus Candida. J. Bacteriol. 79:677-681. Control antisera were alsoraised against S. cerevisiae transformed with the empty plasmid. Allyeast cells were grown in galactose to induce expression of the ALSgenes. Before being tested in the adherence experiments, the serum washeat-inactivated at 56 C to remove all complement activity.

Sera from immunized rabbits were absorbed with whole cells of S.cerevisiae transformed with empty plasmid to remove antibodies that arereactive with components of the yeast other than ALS1 protein. The titerof the antisera was determined by immunofluorescence using S. cerevisiaethat express the ALS1 gene. FITC-labeled anti-rabbit antibodies werepurchased from commercial sources (Southern Biotechnology, Inc).Affinity-purified secondary antibodies were essential because manycommercially available sera contain antibodies reactive with yeastglucan and mannan. The secondary antibodies were pretested using Candidaalbicans as well as S. cerevisiae transformed with the plasmid and wereabsorbed as needed to remove any anti-S. cerevisiae or anti-Candidaantibodies. Negative controls were 1) preimmune serum, 2) S. cerevisiaetransformed with the empty plasmid, and 3) S. cerevisiae transformedwith the ALS gene but grown under conditions that suppress expression ofthe ALS gene (glucose).

In addition to the above experiments, Western blotting was used toprovide further confirmation that an antiserum binds specifically to theALS1 protein against which it was raised. S. cerevisiae transformed withthe ALS1 were grown under inducing conditions and their plasma membraneswere isolated by standard methods. Panaretou, B. and P. Piper. 1996.Isolation of yeast plasma membranes. p. 117-121. In I. H. Evans. (ed.),Yeast Protocols. Methods in Cell and Molecular Biology. Humana Press,Totowa, N.J. Plasma membranes were also prepared from S. cerevisiaetransformed with the empty plasmid and grown under identical conditions.The membrane proteins were separated by SDS-PAGE and then transferred toPVDF membrane by electroblotting. Harlow, E. and D. Lane. 1988.Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press.After being blocked with nonfat milk, the blot was incubated with theALS antiserum. The preabsorbed antiserum did not react with proteinsextracted from S. cerevisiae containing empty plasmid. This antiserumblocked the adherence of S. cerevisiae pYF5 (a clone that expressesCandida albicans ALS1) to endothelial cells.

Example 5 Antibodies Against Specific ALS Proteins ProphylacticallyProtect Mice from Mucosal and Hematogenously Disseminated CandidalInfections

Antisera that block the adherence of a clone of S. cerevisiaetransformed with an ALS1 were demonstrated to protect mice fromintravenous challenge with Candida albicans. The antisera against theALS proteins were first tested in the murine model of hematogenouslydisseminated candidiasis. Affinity-purified anti-ALS antibodies areeffective in preventing adhesion of yeast cells to various substrates(see Example 3). Affinity-purification is useful in this system becauseantibody doses can be accurately determined. Moreover, theunfractionated antisera will undoubtedly contain large amounts ofantibody directed toward antigens on the S. cerevisiae carrier cells.Many of these anti-Saccharomyces antibodies would likely bind to C.albicans and make interpretation of the results impossible.Additionally, it is quite possible that the procedure used to eluteantibodies from S. cerevisiae that express the ALS protein may alsoelute small amounts of yeast mannan or glucan that could haveadjuvant-like activity. The immunoaffinity-purified antibodies arefurther purified before use. They may also be preabsorbed with mousesplenocytes.

Antibody doses may be administered to cover the range that brackets thelevels of serum antibody that can be expected in most activeimmunization protocols and to cover the range of antibody doses that aretypically used for passive immunization in murine models of candidiasis.See Dromer, F., J. Charreirc, A. Contrepois, C. Carbon, and P. Yeni.1987, Protection, of mice against experimental cryptococcosis byanti-Cryptococcus neoformas monoclonal antibody, Infect. Inimun.55:749-752; Han, Y. and J. E. Cutler. 1995, Antibody response thatprotects against disseminated candidiasis, Infect. Immun. 63:2714-2719;Mukherjee, J., M. D. Scharff, and A. Casadevall. 1992, Protective murinemonoclonal antibodies to Cryptococcus neoformas, Infect. Immun.60:4534-4541; Sanford, J. E., D. M. Lupan, A. M. Schlageter, and T. R.Kozel. 1990, Passive immunization against Cryptococcus neoformas with anisotype-switch family of monoclonal antibodies reactive withcryptococcal polysaccharide, Infect. Inunun. 58:1919-1923. BALB/c mice(female, 7 week old, the NCI) were given anti-ALS that had been absorbedwith mouse splenic cells by an intraperitoneal (i.p.) injection. Controlmice received prebled serum that had been absorbed with mouse speniccells, intact anti-ALS serum, or DPBS, respectively. For thepre-absorption, 2 ml of anti-ALS or prebled sera were mixed with 100 μlof mouse (BALB/c, 7 weeks old female, NCI) splenic cells (app. 9×10⁶cells per ml) at room temperature for 20 minutes. The mixture was washedwith warm sterile DPBS by centrifugation (@ 300×g) for 3 minutes. Thisprocedure was repeated three times. The volume of i.p. injection was 0.4ml per mouse. Four hours later, the mice were challenged with C.albicans (strain CA-1; 5×10⁵ hydrophilic yeast cells per mouse) by i.v.injection. Then, their survival times were measured. See FIG. 6.

Previous studies have shown that antibodies administered via theintraperitoneal route are rapidly (within minutes) and almost completelytransferred to the serum (Kozel and Casadevall, unpublishedobservations). As a control for effects of administering the antibodypreparations, a parallel group of mice were treated with antibodiesisolated from pre-immune serum that has been absorbed with S. cerevisiaetransformed with the ALS gene. The survival time and numbers of yeastper gram of kidney were measured. Again, referring to FIG. 6, miceinfected intravenously with 10⁶ blastopores of ALS1 null mutant had alonger median survival time when compared to mice infected with Candidaalbicans CAI-12 or Candida albicans in which one allele of the ALS1 hadbeen deleted (p=0.003).

The N-terminal portion of Als1p was used to generate a mouse monoclonalanti-Als1p antibody using modification of the method described byBrawner and Cutler (1984). Briefly, 6-week old female BALB/c mice (NCl)were immunized by subcutaneous injection with 125 μg of the purifiedN-terminus of the Als1p in 0.25 ml of complete Freund's adjuvant (GibcoBRL). After 21 days, the mice received a subcutaneous booster injectionof another 125 μg of the purified N-terminus of the Als1p in 0.25 ml ofincomplete Freund's adjuvant. On day 28, the mice sera were assessed foranti-Als1p antibodies using enzyme-linked immunosorbent assay (ELISA)plates coated with the N-terminus of the Als1p. A final boosterinjection of 15 μg of the Als1p N-terminus without adjuvant wasadministered intravenously to mice that tested positive for anti-Als1pantibodies 31 days after the initial immunization, and splenocytes wereprepared for hybridoma production as described previously (Brawner andCutler, 1984). Hybridoma antibody production was determined using ELISAplates coated with the purified N-terminus of the Als1p. One of thehybrids obtained produced antibody that agglutinated C. albicans and wascloned four times by limiting dilution. A hybridoma cell line expressingantibody that binds to the same epitope was developed. This antibodyreacted to S. cerevisiae that overexpressed Als1p, but not to S.cerevisiae transformed with the empty plasmid. The antibody also did notreact with S. cerevisiae overexpressing Als5p, Als6p and was only weaklyreactive against Als1p. ALS3p in C. albicans based upon the failure ofthe MAb to recognize any protein in the als1 null mutant strain upongermination. Heavy- and light chain-specific anti-mouse immunoglobulins(ICN Biomedicals) were used in ELISA to isotype this monoclonalantibody. The monoclonal antibody was isotyped to IgG1 with a kappalight chain. Mice administered monoclonal antibodies against theN-terminal domain of ALS1p exhibit a prophylactic and therapeutic effectagainst disseminated candidiasis.

Example 6 Polynucleotide Vaccination Produces Antibodies In Vivo toAlleviate Disseminated Candidal Infections

In this embodiment, an immunogenic ALS polypeptide is introduced to apatient by delivering an effective amount of pharmaceutically acceptablepolynucleotide coding for the selected immunogenic ALS polypeptidewhereby the polynucleotide is expressed in vivo and the patientgenerates an immune response to the immunogen, thereby immunizing thepatient in an equivalent manner to that demonstrated above for theprotein. For example, immunogenic ALS1 polynucleotide compositions,suitable to be used as vaccines, may be prepared from the ALS genes andvectors as disclosed herein. The vaccine elicits an immune response in asubject which includes the production of anti-ALS antibodies thatexhibit specificities for the selected ALS molecule, and may exhibitsimilar affinities and binding to similar epitopes as the polyclonal andmonoclonal antibodies described herein. Immunogenic compositions,including vaccines, containing the ALS nucleic acid may be prepared asinjectables, in physiologically-acceptable liquid solutions or emulsionsfor polynucleotide administration. The nucleic acid may be associatedwith liposomes, such as lecithin liposomes or other liposomes known inthe art, as a nucleic acid liposome (for example, as described in WO9324640) or the nucleic acid may be associated with an adjuvant.Liposomes comprising cationic lipids interact spontaneously and rapidlywith polyanions, such as DNA and RNA, resulting in liposome/nucleic acidcomplexes that capture up to 100% of the polynucleotide. In addition,the polycationic complexes fuse with cell membranes, resulting in anintracellular delivery of polynucleotide that bypasses the degradativeenzymes of the lysosomal compartment. Published PCT application WO94/27435 describes compositions for genetic immunization comprisingcationic lipids and polynucleotides. Agents which assist in the cellularuptake of nucleic acid, such as calcium ions, viral proteins and othertransfection facilitating agents, may advantageously be used. Bothliquid as well as lyophilized forms that are to be reconstituted willcomprise agents, preferably buffers, in amounts necessary to suitablyadjust the pH of the injected solution.

For any parenteral use, particularly if the formulation is to beadministered intravenously, the total concentration of solutes should becontrolled to make the preparation isotonic, hypotonic, or weaklyhypertonic. Non-ionic materials, such as sugars, are preferred foradjusting tonicity, and sucrose is particularly preferred. Any of theseforms may further comprise suitable formulatory agents, such as starchor sugar, glycerol or saline. The compositions per unit dosage, whetherliquid or solid, may contain from 0.1% to 99% of polynucleotidematerial.

The DNA sequences used in these methods can be those sequences which donot integrate into the genome of the host cell. These may benon-replicating DNA sequences, or specific-replicating sequencesgenetically engineered to lack the genome-integration ability. The nakedALS polynucleotide materials comprise the DNA of SEQ ID NO.:7, 9, 11,13, 15, 17, 19, 21, or 23 or in RNA sequences coding for the ALS1ppolypeptide of SEQ ID NO.:8, 10, 12, 14, 16, 18, 20, 22, or 24 includingconservative substitutions and corresponding polynucleotides encodingsuch analogues or derivatives. With the availability of automatednucleic acid synthesis equipment, both the DNA sequences and thecorresponding RNA sequences can be synthesized directly or derived fromthe native organism.

Where the polynucleotide is to be DNA, promoters suitable for use invarious vertebrate systems are well known. For example, for use inmurine systems, suitable strong promoters include RSV LTR, MPSV LTR,SV40 IEP, and metallothionein promoter. In humans, on the other hand,promoters such as CMV IEP may advantageously be used. When thepolynucleotide is mRNA, it can be readily prepared from thecorresponding DNA in vitro. For example, conventional techniques utilizephage RNA polymerases SP6, T3, or T7 to prepare mRNA from DNA templatesin the presence of the individual ribonucleoside triphosphates. Anappropriate phage promoter, such as a T7 origin of replication site isplaced in the template DNA immediately upstream of the gene to betranscribed. Systems utilizing T7 in this manner are well known, and aredescribed in the literature, e.g., in Current Protocols in MolecularBiology, §3.8 (vol. 1 1988).

To produce the composition for injection, any convenient plasmid vectormay be used, preferably comprising a selectable expression vector andpromoter. Suitable plasmids include pc DNA3 (Invitrogen), pCI (Promega),pCMV-beta galactosidase (Clontech) or pRc/CMV-HBs (S) Davis et al. HumanMolecular Genetics 2:1847-1851. The ALS gene is inserted in the vectorin any convenient manner. The gene may be obtained from Candida genomicDNA and amplified using PCR and the PCR product cloned into the vector.The ALS gene plasmid may be transferred, such as by electroporation,into E. coli for replication therein. Plasmids may be extracted from theE. coli in any convenient manner.

The plasmid containing the ALS gene or specified N-terminal fragment maybe administered in any convenient manner to the host, such asintramuscularly, intranasally, intramusonally, intraperitoneally,transdermally or any selected route that elicits the immune response.DNA immunization with the ALS gene or fragment may elicit both cellularand humoral immune responses and produces significant protectiveimmunity and therapeutic effect to Candida albicans.

As noted above, the ALS gene, gene product or specific antibodies may bemixed with pharmaceutically acceptable excipients which are compatibletherewith. Such excipients may include, water, saline, dextrose,glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. Immunogenic compositions and passivevaccines may be administered parenterally, by injection subcutaneously,intravenously, intradermally or intramuscularly, possibly followingpretreatment of the injection site with a local anesthetic.Alternatively, the immunogenic compositions formed according to thepresent invention, may be formulated and delivered in a manner to evokean immune response at mucosal surfaces. Thus, the immunogeniccomposition may be administered to mucosal surfaces by, for example, thenasal or oral (intragastric) routes. Alternatively, other modes ofadministration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkylene glycols or triglycerides. Oral formulations mayinclude normally employed incipients, such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizethe ALS polypeptide or fragment thereof, and antibodies thereto, and ifneeded, to produce a humoral or cell-mediated immune response. Suitabledosage ranges are readily determinable by one skilled in the art and maybe of the order of about 1 microgram to about 1 mg. Suitable regimes forinitial administration and booster doses are also variable, but mayinclude an initial administration followed by subsequentadministrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host. Avaccine which protects against only one pathogen is a monovalentvaccine. Vaccines which contain antigenic material of several pathogensare combined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

Immunogenicity can be significantly improved if immunogens areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the immunogen locally nearthe site of administration to produce a depot effect facilitating aslow, sustained release of antigen to cells of the immune system.Adjuvants can also attract cells of the immune system to the immunogenand stimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to vaccines. Thus, adjuvants have beenidentified that enhance the immune response to antigens. Some of theseadjuvants are toxic, however, and can cause undesirable side-effects,making them unsuitable for use in humans and many animals. Indeed, onlyaluminum hydroxide and aluminum phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating materialcan provoke potent immune responses to antigens. These include saponinscomplexed to membrane protein antigens to produce immune stimulatingcomplexes (ISCOMS), pluronic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as monophoryl lipid A, QS 21 and polyphosphazene.

The particular examples set forth herein are instructional and shouldnot be interpreted as limitations on the applications to which those ofordinary skill are able to apply this invention. Modifications and otheruses are available to those skilled in the art which are encompassedwithin the spirit and scope of the following claims.

I claim:
 1. A monoclonal antibody against an ALS1 protein thatspecifically binds an epitope in an N-terminal domain and which inhibitsadherence of Candida albicans to endothelial cells.
 2. The monoclonalantibody of claim 1 which inhibits filamentation of Candida albicans. 3.The monoclonal antibody of claim 1 which is isotype IgG.
 4. Themonoclonal antibody of claim 1 which preferentially reacts with aSaccharomyces cerevisae cell line that overexpresses ALS.
 5. Themonoclonal antibody of claim 1 encoded by a human immunoglobulin gene ina transgenic mouse.
 6. A hybridoma cell line that produces the antibodyof claim
 1. 7. A pharmaceutical composition comprising the monoclonalantibody of claim
 1. 8. A vaccine comprising: a pharmaceuticalcomposition comprising an isolated and substantially pure cell surfaceadhesin protein obtained from a Candida strain.
 9. The vaccine of claim8, wherein the cell surface adhesin protein is Candida albicans ALS1adhesion protein (Als1p).
 10. The vaccine of claim 8, wherein theprotein is a fragment encompassing an N-terminal domain that contains anadhesin binding site for adherence of Candida albicans to endothelialcells.
 11. The vaccine of claim 8, wherein said cell surface adhesinprotein is derived from a Candidal strain selected from the groupconsisting of Candida krusei, Candida dublinoensis, Candidaguilliermondii, Candida tropicalis, and Candida parapsilosis.
 12. Amethod for treatment or prevention of disseminated candidiasis,comprising: administering as a vaccine a pharmaceutical compositioncomprised of a pharmacologically effective amount of an ALS adhesionprotein of Candida albicans in an amount that generates a therapeutic orprophylactic immune response characterized by production of antibodiesthat inhibit adherence of Candida albicans to endothelial cells.
 13. Themethod of claim 12, wherein the administering step is comprised ofadministering a substantially pure formulation of an N-terminal domainof the ALS adhesin protein.
 14. The method of claim 13, wherein theadministering step further comprises the step of administering animmunostimulatory adjuvant.
 15. A composition for the treatment orprophylaxis of candidiasis comprising a pharmaceutical compositioncomprising a fungicidal effective amount of polyclonal immunoglobulinscontaining antibodies that specifically bind the ALS adhesin protein.16. The composition of claim 16, wherein the polyclonal immunoglobulinsare specific to an N-terminal region.
 17. A method for the treatment orprophylaxis of candidiasis comprising administering to a patient afungicidal effective amount of polyclonal immunoglobulins containingantibodies against an adhesin protein.
 18. The method of claim 17,wherein the polyclonal immunoglobulins are specific to an N-terminalregion of ALS1p.
 19. A pharmaceutical composition comprising an ALSpolynucleotide characterized by in vivo expression of an immunogenic ALSpolypeptide that exhibits an immune response producing antibodiesspecific to the ALS polypeptide.
 20. The pharmaceutical composition ofclaim 19 wherein the ALS1 polynucleotide encodes an N-terminal domain ofthe ALS polypeptide.
 21. The pharmaceutical composition of claim 19wherein the antibodies inhibit adherence of Candida albicans toendothelial cells.